The prokaryotic mRNA ribosome binding site (RBS) usually contains part or all of a polypurine domain UAAGGAGGU known as the Shine-Dalgarno (SD) sequence found just 5' to the translation initiation codon. It is now clear that the SD sequence is important for identification of the translation initiation site on the mRNA by the ribosome, and that as a result, the spacing between the SD and the initiation codon strongly affects translational efficiency (1). It is not as clear, however, whether there is a unique optimal spacing. Complications involving the definition of the spacing as well as secondary structures have obscured matters. We thus undertook a systematic study by inserting two series of synthetic RBSs of varying spacing and SD sequence into a plasmid vector containing the chloramphenicol acetyltransferase gene. Care was taken not to introduce any secondary structure. Measurements of protein expression demonstrated an optimal aligned spacing of 5 nt for both series. Since aligned spacing corresponds naturally to the spacing between the 3'-end of the 16S rRNA and the P-site, we conclude that there is a unique optimal aligned SD-AUG spacing in the absence of other complicating issues.

Several electrophoretic and chromatographic systems have been investigated and compared for sequence analysis of oligodeoxyribonucleotides. Three systems were found to be useful for the separation of a series of sequential degradation products resulting from a labeled oligonucleotide: (I) 2-D electrophoresisdagger; (II) 2-D PEI-cellulose; and (III) 2-D homochromatography. System (III) proved generally most informative regardless of base composition and sequence. Furthermore, only in this system will the omission of an oligonucleotide in a series of oligonucleotides be self-evident from the two-dimensional map. The sequence of up to fifteen nucleotides can be determined solely by the characteristic mobility shifts of its sequential degradation products distributed on the two-dimensional map. With this method, ten nucleotides from the double-stranded region adjacent to the left-hand 3'-terminus and seven from the right-hand 3'-terminus of bacteriophage lambda DNA have been sequenced. Similarly, nine nucleotides from the double-stranded region adjacent to the left-hand 3'-terminus and five nucleotides from the right-hand terminus of bacteriophage phi80 DNA have also been sequenced. The advantages and disadvantages of each separation system with respect to sequence analysis are discussed.

Terminal deoxynucleotidyl transferase, which requires a single-stranded DNA primer under the usual assay conditions, can be made to accept double-stranded DNA as primer for the addition of either rNMP or dNMP, if Mg+2 ion is replaced by Co+2 ion. The priming efficiency in the presence of (C leads to) CO+2 ion with respect to initial rate tested with 2 single-stranded primer, is 5-6 fols higher than that observed with Mg+2 ion. In the presence of Co+2 ion, the primer specificity is altered so that all forms of duplex DNA molecules can be labeled at their unique 3' -ends regardless of whether such ends are staggered or even. Thus, using ribonucleotide incorporation, we have for the first time employed this reaction for sequence analysis of duplex DNA fragments generated by restriction endonuclease cleavages. Furthermore, by using Co+2 ion, it is possible to add a long homopolymer tract of deoxyribonucleotides to the 3'-terminus of double-stranded DNA. Therefore, without prior treatment with lambda exonuclease to expose the 3' terminus as single-stranded primer, this reaction now permits insertion of homopolymer tails at the 3'-ends of all types of DNA molecules for the purpose of in vitro construction of recombinant DNA.

Phycocyanin (PC) is a light-harvesting protein common to blue-green and red algae. We have isolated the genes for the two apoprotein subunits, a and f, of PC from the blue-green alga Agmenellum quadruplicatum In most photosynthetic organisms the major light-harvesting pigments are cyclic tetrapyrroles bound noncovalently to polypeptides intrinsic to the photosynthetic membrane. Blue-green algae (cyanobacteria) and red algae follow this pattern but, in addition, have large amounts of antenna pigments in the form of linear tetrapyrroles covalently bound to water-soluble polypeptides. These proteins, termed phycobiliproteins, occur as three major types: phycoerythrin, phycocyanin (PC), and allophycocyanin (1). Each phycobiliprotein consists of two subunits, a and /3, which contain characteristic numbers and types of chromophores. Phycobiliproteins aggregate to form complexes called phycobilisomes (1). The assembly of phycobilisomes is mediated by nonpigmented linker polypeptides. These linkers also alter the spectral properties of phycobiliproteins so as to ensure an efficient transfer of absorbed light energy to the membrane (2). Phycobilisomes, in turn, are found attached to the outer surface of the thylakoid membrane, perhaps in association with photosystem 11 (3).Our aim is to describe the structure of the phycobilisome and the regulation of the genes encoding its components.These studies may also shed light on the evolution of Ohycobiliprotein genes. As a first step, we have cloned and sequenced the genes encoding the a and ,8 subunit apoproteins of PC from the blue-green alga Agmenellum quadruplicatum. A preliminary report of these results has been presented (4). MATERIALS AND METHODSDNA purification. A. quadruplicatum strain PR-6 was cultivated axenically in medium A with NaNO3 (1 mg/ml) as described (5). Cells were harvested before reaching a density of 5 x 107 cells per ml, washed in 10% sucrose/50 mM Tris HCl, pH 8.0/100 mM Na2EDTA, and stored at -80°C. Lysis was achieved by thawing, adding egg-white lysozyme to a final concentration of 10 mg/ml, incubating at 37°C for 30 min, and adding N-lauroyl sarcosine (10% wt/vol stock solution) to a final concentration of 1%. An equal amount (wt/vol) of CsCl was added and DNA was purified by buoyant-density centrifugation. DNA-containing fractions were recentrifuged in the presence of ethidium bromide at 150 ,ug/ml. The dye was removed by n-butanol extraction and the DNA was dialyzed against 10 mM Tris-HCl, pH 8.0/1 mM Na2EDTA.RNA Purification. An exponentially growing culture of A. quadruplicatum was harvested, resuspended in the original volume of fresh medium A lacking nitrate, and incubated with aeration and illumination as before. The A620/A680 ratio decreased from 0.9 to 0.3 within 24 hr, whereupon the cells were harvested, resuspended in medium A with nitrate (1 mg/ml) and incubated as before. After 8 hr, cells were harvested by centrifugation and resuspended in 10 mM Tris HCl, pH 8.0/1 mM Na2EDTA. Cells were lysed by passage through a French pressu...

The genes for the a-and a-subunit apoproteins of allophycocyanin (AP) were isolated from the cyanelle genome of Cyanophora paradoxa and subjected to nucleotide sequence analysis. The AP a-subunit apoprotein gene was localized to a 7.8-kilobase-pair Pst I restriction fragment from cyanelle DNA by hybridization with a tetradecameric oligonucleotide probe. Sequence analysis using that oligonucleotide and its complement as priners for the dideoxy chain-termination sequencing method confirmed the presence of both AP viand ,8-subunit genes on this restriction fragment. Additional oligonucleotide primers were synthesized as sequencing progressed and were used to determine rapidly the nucleotide sequence of a 1336-base-pair region of this cloned fragment. This strategy allowed the sequencing to be completed without a detailed restriction map and without extensive and timeconsuming subcloning. The sequenced region contains two open reading frames whose deduced amino acid sequences are 81-85% homologous to cyanobacterial and red algal AP subunits whose amino acid sequences have been determined. The two open reading frames are in the same orientation and are separated by 39 base pairs. AP a is 5' to AP fi and both coding sequences are preceded by a polypurine, ShineDalgarno-type sequence. Sequences upstream from AP a closely resemble the Escherichia coli consensus promoter sequences and also show considerable homology to promoter sequences for several chloroplast-encoded psbA genes. A 56-base-pair palindromic sequence downstream from the AP fi gene could play a role in the termination of transcription or translation. The allophycocyanin apoprotein subunit genes are located on the large single-copy region of the cyanelle genome.